Changes associated with aging and replicative senescence in the regulation of transcription factor nuclear factor-κB

Abstract

Both the aging of animals and the senescence of cultured cells involve an altered pattern of gene expression, suggesting changes in transcription factor regulation. We studied age-related changes in transcription factors nuclear factor (NF)-κB, activator protein factor-1 (AP-1) and Sp-1 by using electrophoretic mobility shift binding assays; we also analysed changes in the protein components of NF-κB complex with Western blot assays. Nuclear and cytoplasmic extracts were prepared from heart, liver, kidney and brain of young adult and old NMRI mice and Wistar rats as well as from presenescent, senescent and simian virus 40-immortalized human WI-38 fibroblasts. Aging of both mice and rats induced a strong and consistent increase in the nuclear binding activity of NF-κB factor in all tissues studied, whereas those of AP-1 and Sp-1 decreased, e.g. in liver. Protein levels of p50, p52 and p65 components of the NF-κB complex did not show any age-associated changes in the cytoplasmic fraction but in the nuclear fraction the level of p52 strongly increased in heart and liver during aging. The protein levels of inhibitory IκB-α and Bcl-3 components were not affected by aging in any of the tissues studied. Replicative cellular senescence of human WI-38 fibroblasts induced a strong decrease in nuclear NF-κB, AP-1 and Sp-1 binding activities. Protein levels of p50 and p52 components of NF-κB complex were decreased in the nuclear fraction of senescent WI-38 fibroblasts but in the cytoplasm of senescent fibroblasts the level of p65 protein was increased. Cellular senescence also slightly decreased the protein levels of IκB-α and Bcl-3. Transfection assays with NF-κB-enhancer-driven chloramphenicol acetyltransferase reporter gene showed a significant down-regulation of NF-κB promoter activity in senescent WI-38 fibroblasts. Results suggest that the aging process might be regulated differently in tissues and cultured fibroblasts, perhaps reflecting differences between mitotic and post-mitotic cells. In tissues, aging seems to involve specific changes in the regulation of NF-κB components and perhaps also changes in the DNA-binding affinities of the NF-κB complex.